Oncolytic vaccinia virus combination cancer therapy

ABSTRACT

Embodiments of the invention are directed methods that include a thymidine kinase deficient vaccinia virus. The methods include evaluating a tumor for reperfusion after treatment with vaccinia virus and administering an anti-angiogenic agent if reperfusion is detected.

This application is a national stage filing of International applicationNo. PCT/US2010/048829 filed Sep. 14, 2009 (pending), which is anon-provisional application of U.S. Provisional Patent applications61/242,238 filed Sep. 14, 2009 and 61/244,250 filed Sep. 21, 2009. Thisapplication claims priority to each application referenced above and theentire contents of each of the above referenced disclosures areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of oncology andvirology. More particularly, it concerns poxviruses, specificallyincluding oncolytic vaccinia viruses suitable for the treatment ofcancer and their use in combination with anti-angiogenic agents.

II. Background

Normal tissue homeostasis is a highly regulated process of cellproliferation and cell death. An imbalance of either cell proliferationor cell death can develop into a cancerous state (Solyanik et al., 1995;Stokke et al., 1997; Mumby and Walter, 1991; Natoli et al., 1998;Magi-Galluzzi et al., 1998). For example, cervical, kidney, lung,pancreatic, colorectal, and brain cancer are just a few examples of themany cancers that can result (Erlandsson, 1998; Kolmel, 1998; Mangrayand King, 1998; Mougin et al., 1998). In fact, the occurrence of canceris so high that over 500,000 deaths per year are attributed to cancer inthe United States alone.

Currently, there are few effective options for the treatment of manycommon cancer types. The course of treatment for a given individualdepends on the diagnosis, the stage to which the disease has developedand factors such as age, sex, and general health of the patient. Themost conventional options of cancer treatment are surgery, radiationtherapy and chemotherapy. Surgery plays a central role in the diagnosisand treatment of cancer. Typically, a surgical approach is required forbiopsy and to remove cancerous growth. However, if the cancer hasmetastasized and is widespread, surgery is unlikely to result in a cureand an alternate approach must be taken.

Replication-selective oncolytic viruses hold promise for the treatmentof cancer (Kirn et al., 2001). These viruses can cause tumor cell deaththrough direct replication-dependent and/or viral geneexpression-dependent oncolytic effects (Kirn et al., 2001). In addition,viruses are able to enhance the induction of cell-mediated antitumoralimmunity within the host (Todo et al., 2001; Sinkovics et al., 2000).These viruses also can be engineered to expressed therapeutic transgeneswithin the tumor to enhance antitumoral efficacy (Hermiston, 2000).However, major limitations exist to this therapeutic approach as well.

Therefore, more additional therapies for the treatment of cancer areneeded. The use of oncolytic viruses presents a potential area fordevelopment.

SUMMARY OF THE INVENTION

In vitro studies have indicated that sorafenib and similar kinaseinhibitors suppress the effectiveness of poxvirus, vaccinia virus andparticularly JX-594 when used in combination on cultured cell lines.Contrary to the in vitro findings, preclinical efficacy models haveshown that combining sorafenib and JX-594 actually shows better efficacythan either agent alone. Thus, various aspects of the invention aredirected to the application of these unexpected findings as in vivotherapy using a combination of poxvirus, vaccinia virus, or JX-594 virusand anti-angiogenic agents, such as kinase inhibitors, sorafenib,sutent, or similar compounds.

Embodiments of the invention are directed to methods for treating cancerin a subject previously administered a poxvirus therapy comprisingadministering an effective amount of an anti-angiogenic agent. Incertain aspects it is determined that the tumor being treated isundergoing re-vascularization. In a further aspect the poxvirus is avaccinia virus. In still a further aspect the vaccinia virus is avaccinia virus expressing GM-CSF. Alternatively the vaccinia virus lacksa functional thymidine kinase gene. In certain aspects the vacciniavirus is JA-594.

Certain embodiments are directed to potentiating anti-angiogenictherapy, particularly those for which a patient has failed, hasdeveloped a tolerance, does not respond, or partially responds. As usedherein, the term “potentiate”, “potentiating”, “therapy potentiating”,“therapeutic effect is potentiated”, and “potentiating the therapeuticeffects” is defined herein as producing one or more of the followingphysiological effects: the increase or enhancement of the cytotoxicactivity of therapeutic agents by acting in an additive or synergisticcytotoxic manner with the therapeutic agents; sensitizing cancer cellsor a tumor to the anti-cancer activity of therapeutic agents; and/orrestoring anti-angiogenic effectiveness of a therapy or sensitivity of atumor to the therapy. Embodiments of the invention includeanti-angiogenic agents as therapeutic agents for the treatment ofcancer. In certain aspects methods of potentiating anti-angiogenictherapy include administering a poxvirus to a patient that isinsensitive to, developed a tolerance for, or is not sufficientlyresponding to anti-angiogenic therapy in an amount that potentiates thetherapeutic efficacy of the anti-angiogenic therapy. In further aspectsthe anti-angiogenic therapy is a kinase inhibitor, sorafenib, sutent, orsimilar compound. Methods of the invention can also include identifyinga patient that is resistant or non-responsive or has cancer recurrenceafter anti-angiogenic therapy. In certain aspects a sensitizing amountof poxvirus, vaccinia virus, or JX-594 virus is administered to apatient that is resistant, tolerant, or insensitive to anti-angiogenictherapy (anti-angiogenic kinase inhibitors, sorafenib, sutent or similarcompounds). A sensitizing amount is an amount sufficient render a tumornot showing a therapeutic response to a treatment—as determined by aphysician or scientist—capable of responding to the same or similartherapy.

“Therapy resistant” cancers and tumors refers to cancers that havebecome resistant to anti-angiogenic cancer therapies. “Therapysensitive” cancers are responsive (clinical parameters of response aredetectable or measurable, such as tumor growth reduction, tumornecrosis, tumor shrinkage, tumor vascular shutdown and the like) totherapy. One of skill in the art will appreciate that some cancers aretherapy sensitive to particular agents but not to others.

In certain aspects the anti-angiogenic agent is a kinase inhibitor. Inother aspects the kinase inhibitor inhibits the Raf kinase pathway. In aparticular aspect the kinase inhibitor is sorafenib, sutent, or similaranti-angiogenic kinase inhibitor.

Certain embodiments are directed to methods further comprisingdetermining if a tumor is undergoing re-vascularization. In certainaspects re-vascularization is determined by non-invasive imaging of thetumor, for example, magnetic resonance imaging (MRI). In certain aspectsthe magnetic resonance imaging is dynamic contrast-enhanced MRI(DCE-MRI).

In certain aspects of the methods the anti-angiogenic agent isadministered at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks,including all values and ranges there between, after the first, second,third, fourth, fifth or more vaccinia virus administration.

In certain aspects the tumor is a brain tumor, a head & neck cancertumor, an esophageal tumor, a skin tumor, a lung tumor, a thymic tumor,a stomach tumor, a colon tumor, a liver tumor, an ovarian tumor, auterine tumor, a bladder tumor, a testicular tumor, a rectal tumor, abreast tumor, a kidney tumor, or a pancreatic tumor. In a further aspectthe tumor is a hepatocellular carcinoma or a colorectal cancer.

In certain aspects the methods further comprising first administering tothe subject the poxvirus, vaccinia virus, or JX-594 viral therapy. In afurther aspect the viral therapy can be administered by injection into atumor mass or by intravascular administration. In a particular aspectthe virus is injection into tumor vasculature. In certain aspects theviral therapy can be administered via multiple modalities, e.g.,intravascular and intratumoral, etc.

Certain embodiments are directed to methods for treating a hepatic tumoror metastatic tumor in the liver or other organ of a patient comprisingadministering sorafenib to the tumor, wherein the tumor was previoustreated with a poxvirus, vaccinia virus, or JX-594 virus. Methods of theinvention can also include determining if the tumor is undergoingreperfusion.

Certain aspects are directed to methods of treating a hepatic tumor,either primary or a metastatic tumor (a metastatic tumor being a tumorthat originates in an organ or tissue distal from the location in whichit is treated) comprising administering an effective amount of apoxvirus, a vaccinia virus, or a JX-594 virus, and administering ananti-angiogenic agent. In certain aspects the anti-angiogenic agent willbe a kinase inhibitor. In further aspects the kinase inhibitor issorafenib or sutent.

Certain embodiments are directed to methods of treating a hepatic tumorcomprising administering an effective amount of a poxvirus, a vacciniavirus, or a JX-594 virus, wherein the tumor will be evaluated forreperfusion and determined to be a candidate for sorafenib therapy ifthe tumor is undergoing reperfusion.

In still further aspects are directed to methods of treating a patienthaving a tumor comprising (a) evaluating a tumor that has been treatedwith an anti-cancer therapy by non-invasive imaging of the tumor todetect reperfusion; and (b) administering an effective amount ofanti-angiogenic agent, e.g., sorafenib or sutent, or similar kinaseinhibitor, to a tumor in which reperfusion is detected or suspected.Imaging is not required for treating the a tumor with the combination ofJX-594 and an anti-angiogenic such as sorafenib or sutent or similarkinase inhibitor.

JX-594 is a targeted oncolytic poxvirus designed to selectivelyreplicate in and destroy cancer cells. Direct oncolysis plus granulocytemacrophage—colony stimulating factor (GM-CSF) expression also stimulatestumor vascular shutdown in tumors.

Certain embodiments of the invention are directed to methods thatinclude administration of a thymidine kinase deficient vaccinia virus.In certain aspects, the methods include administering to the subject aTK-deficient, GM-CSF-expressing, replication-competent vaccinia virusvector (e.g., JX-594) in an amount sufficient to induce oncolysis ofcells in the treated tumor or other tumors distal from theadministration site. The administration of vaccinia virus can befollowed by administration an anti-angiogenic agent, such as ananti-angiogenic tyrosine kinase inhibitor.

The tyrosine kinase inhibitor can be selected from the group consistingof sunitinib (SU1 1248; Sutent®), SU5416, SU6668, vatalanib(PTK787/ZK222584), AEE788, ZD6474, ZD4190, AZD2171, GW786034, sorafenib(BAY 43-9006), CP-547,632, AG013736, YM-359445, gefitinib (Iressa®),erlotinib (Tarceva®), EKB-569, HKI-272, and CI-1033. Sorafenib (Nexavar,Bayer), is a drug approved for the treatment of primary kidney cancer(advanced renal cell carcinoma) and advanced primary liver cancer(hepatocellular carcinoma). Sorafenib is a small molecular inhibitor ofseveral Tyrosine protein kinases. Sorafenib targets the Raf/Mek/Erkpathway (MAP Kinase pathway). Therefore, other kinase inhibitors thattarget this pathway are also contemplated as being useful in combinationwith JX-594.

In certain aspects, the subject is administered at least 1×10⁷, 1×10⁸,2×10⁸, 5×10⁸, 1×10⁹2×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹, 1×10¹²,5×10¹² or more viral particles or plaque forming units (pfu), includingthe various values and ranges there between. The viral dose can beadministered in 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, 1000 ormore milliters, including all values and ranges there between. In oneaspect, the dose is sufficient to generate a detectable level of GM-CSFin serum of the patient, e.g., at least about, at most about or about 2,5, 10, 40, 50, 100, 200, 500, 1,000, 5,000, 10,000, 15,000 to 20,000pg/mL, including all values and ranges there between. It is contemplatedthat a single dose of virus refers to the amount administered to asubject or a tumor over a 0.1, 0.5, 1, 2, 5, 10, 15, 20, or 24 hourperiod, including all values there between. The dose may be spread overtime or by separate injection. Typically, multiple doses areadministered to the same general target region, such as in the proximityof a tumor or in the case of intravenous administration a particularentry point in the blood stream or lymphatic system of a subject. Incertain aspects, the viral dose is delivered by injection apparatuscomprising a needle providing multiple ports in a single needle ormultiple prongs coupled to a syringe, or a combination thereof. In afurther aspect, the vaccinia virus vector is administered 2, 3, 4, 5, ormore times. In still a further aspect, the vaccinia virus isadministered over 1, 2, 3, 4, 5, 6, 7 or more days or weeks.

In certain embodiments the subject is a human. The subject may beafflicted with cancer and/or a tumor. In certain embodiments the tumormay be non-resectable prior to treatment and resectable followingtreatment. In certain aspects the tumor is located on or in the liver.In other aspects, the tumor can be a brain cancer tumor, a head and neckcancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lungcancer tumor, a thymic cancer tumor, a stomach cancer tumor, a coloncancer tumor, a liver cancer tumor, an ovarian cancer tumor, a uterinecancer tumor, a bladder cancer tumor, a testicular cancer tumor, arectal cancer tumor, a breast cancer tumor, or a pancreatic cancertumor. In other embodiments the tumor is a bladder tumor. In stillfurther embodiments the tumor is melanoma. The tumor can be a recurrent,primary, metastatic, and/or multi-drug resistant tumor. In certainembodiments, the tumor is a hepatocellular tumor or a metastasized tumororiginating from another tissue or location. In certain aspects thetumor is in the liver.

In certain aspects the patient is monitored for tumor reperfusion. Incertain aspects monitoring or evaluating the patient will be bynon-invasive or minimally invasive imaging, e.g., magnetic resonanceimaging. If reperfusion is detected or suspect a patient can beadministered an anti-angiogenic agent, such as an anti-angiogenictyrosine kinase inhibitor. In certain aspects, the tyrosine kinaseinhibitor is sorafenib or similar agent. The anti-angiogenic agent canbe administer at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more weeks after poxvirus virus therapy.

In certain aspects, the method further comprises administering to thesubject an additional cancer therapy. The additional cancer therapy canbe chemotherapy, biological therapy, radiotherapy, immunotherapy,hormone therapy, anti-vascular therapy, cryotherapy, toxin therapyand/or surgery, including combinations thereof. In still a furtheraspect, surgery includes the transarterial chemoembolization (TACEprocedure, see Vogl et al., European Radiology 16(6):1393, 2005). Themethod may further comprise a second administration of the vacciniavirus vector. Methods of the invention can further comprise assessingtumor cell viability before, during, after treatment, or a combinationthereof. In certain embodiments the virus is administeredintravascularly, intratumorally, or a combination thereof. In a furtheraspect administration is by injection into a tumor mass. In still afurther embodiment, administration is by injection into or in the regionof tumor vasculature. In yet a further embodiment, administration is byinjection into the lymphatic or vasculature system proximal to thetumor. In certain aspects the method includes imaging the tumor prior toor during administration. In certain aspects, a patient is or is notpre-immunized with a vaccinia virus vaccine. In a further aspect, thesubject can be immunocompromised, either naturally or clinically.

In certain aspects, the virus is administered in an amount sufficient toinduce cell or cancer cell death or necrosis in at least 15% of cells inan injected tumor, in at least 20% of cells in an injected tumor, in atleast 30% of cells in an injected tumor, in at least 30% of cells in aninjected tumor, in at least 40% of cells in an injected tumor, in atleast 50% of cells in an injected tumor, in at least 60% of cells in aninjected tumor, in at least 70% of cells in an injected tumor, in atleast 80% of cells in an injected tumor, or in at least 90% of cells inan injected tumor.

In a further aspect of the invention, the methods can excludepre-treatment of a subject with a vaccinia vaccine, e.g., a subject neednot be vaccinated 1, 2, 3, 4, 5, or more days, weeks, months, or yearsbefore administering the therapy described herein. In some aspects,non-injected tumors or cancer will be infected with the therapeuticvirus, thus treating a patient by both local administration and systemicdissemination.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. The embodiments in the Example section are understood to beembodiments of the invention that are applicable to all aspects of theinvention.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. JX-594 replication is inhibited in the presence of sorafenib invitro. At various multiplicities of infection (MOI), JX-594 alone, orwith 10 μM sorafenib, was added to PLC/PRF/5 cells. After 24 hoursinfection, cells and supernatants were collected for titration by plaqueassay on Vero cells.

FIG. 2. Sorafenib Inhibits JX-594 Plaque Formation and Replication.JX-594 was allowed to infect monolayers of A2780 or HepG2 cells in theabsence or presence of increasing concentrations of sorafenib. Toppanels show experiments measuring plaque formation on the originalmonolayer, and the production of new viral particles (burst). Bottompanel shows that concentrations below cytotoxic levels are effective atinhibiting viral replication. The data are expressed as percent ofcontrol (no JX-594, no sorafenib). Error bars are standard deviation ofreplicates

FIG. 3. Combination therapy with sorafenib enhances JX-594 efficacyagainst CT26 subcutaneous solid tumors. Top panel shows the study designof a combination efficacy preclinical study in mice with CT2subcutaneous tumors. Kaplan-Meier survival curves are shown for eachcondition in the middle panel. Effects on tumor volume are shown in thelower panel.

FIG. 4. Combination therapy with sorafenib enhances JX-594 efficacy inthe murine B16 metastatic melanoma model. Top panel shows the studydesign of a combination efficacy preclinical study in B16 murine tumormodel. Bottom panel shows average number of lung metastases thatdeveloped in each group.

FIG. 5 JX-594 followed by Sorafenib shows superior efficacy in HCCxenograft model. Chart describes treatment schedule for each group.Graph plots average tumor size (mm3) for each group over time (bars arestandard error of the mean).

FIG. 6 Anti-vascular effects of JX-594 followed by sorafenib. Top panelsshow study design of a preclinical study of JX-594 followed by Sorafenibin HepG2 xenograft model. Bottom panel shows average number of vesselsin tumors (three 200× fields were counted). Error bars are standarderror.

FIG. 7 DCE-MRI scan of HCC patient showing vascular shutdown.

FIG. 8 DCE-MRI scans showing response in sites distant to intratumoralinjection cites.

FIG. 9 DCE-MRI scans showing response in sites distant to intratumoralinjection cites.

FIG. 10 Illustrates a summary of patient responses, including Choiassessment.

FIG. 11 Patient 1702—DCE-MRI Images 4 weeks and 8 weeks Post-SorafenibTreatment (3 different planes are shown, with 4 week and 8 week imagesof each plane).

FIG. 12 Patient 1705—DCE-MRI images before and 5 days post-JX-594treatment: DCE-MRI images before and 4 weeks post-Sorafenib treatment.

FIG. 13 Patient 1712—DCE-MRI images before and 4 weeks post-Sorafenibtreatment.

FIG. 14 Patient 11301—assessment of patient with renal cell carcinomametastasis to liver.

FIG. 15 Illustration of tumor stabilization and decreased enhancementobserved with sequential JX-594 and sorafenib (Patient 1705).

FIG. 16 Illustrates significant necrosis induction in patient treatedwith JX-594 followed by sorafenib (Patient 1705).

FIG. 17 Illustrates reduced viable tumor volume following sequentialtherapy with JX-594 and sorafenib.

FIG. 18 DCE-MRI scans of Patient having hepatocellular carcinoma and wasenrolled in a Phase 2 Clinical Trial of JX-594 showed loss of perfusion10 days after sorafenib initiation in a non-injected extahepatic tumor.After completing JX-594 administration (one intravenous and twointratumoral doses) patient received sorafenib.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the use of oncolytic poxviruses for thetreatment of cancer. In particular, the use of a vaccinia virusexpressing GM-CSF to achieve a particular degree of oncolysis isdescribed. In another embodiment, a vaccinia virus can be used in atreatment regime that is more effective at treating vascularized orvascularizing tumors. A particular regime is the use of ananti-angiogenic agent after treatment with an oncolytic vaccinia virus.In certain aspects the treatment regime includes imaging the tumor toassess reperfusion that results from re-vacularization of the tumorafter the vaccinia virus induce vascular collapse of the tumor. Incertain aspects, the vaccinia virus is the JX-594 virus (TK minus,GM-CSF expressing vaccinia virus).

I. Treatment Regimens and Pharmaceutical Formulations

In an embodiment of the present invention, a method of treatment for ahyperproliferative disease, such as cancer, by the delivery of anoncolytic vaccinia virus, such as JX-594, is contemplated.

The methods include administrations of an effective amount of apharmaceutical composition comprising an oncolytic vaccinia virus or ananti-angiogenic agent. A pharmaceutically effective amount is defined asthat amount sufficient to induce oncolysis—the disruption or lysis of acancer cell—and/or the inhibition of vascularization or destruction ofneo-vasculature of tumors. The term includes the slowing, inhibition, orreduction in the growth or size of a tumor and includes the eradicationof the tumor in certain instances. In certain aspects an effectiveamount of vaccinia virus results in systemic dissemination of thetherapeutic virus to tumors, e.g., infection of non-injected tumors.

A. Combination Treatments

The compounds and methods of the present invention may be used in thecontext of hyperproliferative diseases/conditions, including cancer, andmay be used in a particular order of administration with various timegaps between administration. In order to increase the effectiveness of atreatment with the compositions of the present invention, such as aGM-CSF-expressing vaccinia virus, it is desirable to combine thesecompositions with anti-angiogenic agents and other agents effective inthe treatment of cancer. For example, the treatment of a cancer may beimplemented with therapeutic compounds of the present invention incombination with anti-angiogenic agents.

Various combinations may be employed; for example, a poxvirus, such asvaccinia virus JX-594, is “A” and the secondary anti-angiogenic therapyis “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/BB/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the poxvirus/vaccinia vectors of the present inventionto a patient will follow general protocols for the administration ofthat particular therapy, taking into account the toxicity, if any, ofthe poxvirus treatment. After treatment with vaccinia virus ananti-angiogenic agent is administered to effectively treatneo-vascularization or reperfusion of the vaccinia virus treated tumor.It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedcancer or tumor cell therapy.

An “anti-angiogenic” agent is capable of negatively affectingangiogenesis in a tumor, for example, by killing cells, inducingapoptosis in cells, reducing the growth rate of cells involved inangiogenesis and effectively reducing the blood supply to a tumor orcancer cell. Examples of anti-angiogenic agents include, but are notlimited to, sorafenib, sutent and similar compounds, retinoid acid andderivatives thereof, 2-methoxyestradiol, ANGIOSTATIN™, ENDOSTATIN™,suramin, squalamine, tissue inhibitor of metalloproteinase-1, tissueinhibitor of metalloproteinase-2, plasminogen activator inhibitor-1,plasminogen activator inhibitor-2, cartilage-derived inhibitor,paclitaxel, platelet factor 4, protamine sulphate (clupeine), sulphatedchitin derivatives (prepared from queen crab shells), sulphatedpolysaccharide peptidoglycan complex (sp-pg), staurosporine, modulatorsof matrix metabolism, including for example, proline analogs((I-azetidine-2-carboxylic acid (LACA), cishydroxyproline,d,I-3,4-dehydroproline, thiaproline, alpha-dipyridyl,beta-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone; methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin,β-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodiumthiomalate, d-penicillamine (CDPT), beta-1-anticollagenase-serum, alpha2-antiplasmin, bisantrene, lobenzarit disodium,n-2-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”,thalidomide; angiostatic steroid, carboxynaminolmidazole;metalloproteinase inhibitors such as BB94. Other anti-angiogenesisagents include antibodies, for example, monoclonal antibodies againstthese angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms,VEGF-C, HGF/SF and Ang-1/Ang-2. Ferrara N. and Alitalo, K. “Clinicalapplication of angiogenic growth factors and their inhibitors” (1999)Nature Medicine 5:1359-1364. Other anti-angiogenesis agents may includeinhibitors of VEGF transcription.

Angiogenesis, the formation of new blood vessels out of pre-existingcapillaries, is a sequence of events that is of key importance in abroad array of physiologic and pathologic processes. A number ofdiseases are associated with formation of new vasculature. Angiogenesisis an important characteristic of various pathologies, includingpathologies characterized or associated with an abnormal or uncontrolledproliferation of cells such as tumors. Pathologies which involveexcessive angiogenesis include, for example, cancers (both solid andhematologic tumors). Cancer patient can benefit from inhibition ofangiogenesis—tumor vascularization.

Angiogenesis is crucial to the growth of neoplastic tissues. For morethan 100 years, tumors have been observed to be more vascular thannormal tissues. Several experimental studies have suggested that bothprimary tumor growth and metastasis require neovascularization.Pathologic angiogenesis necessary for active tumor growth is generallysustained and persistent, with the initial acquisition of the angiogenicphenotype being a common mechanism for the development of a variety ofsolid and hematopoietic tumor types. Tumors that are unable to recruitand sustain a vascular network typically remain dormant as asymptomaticlesions in situ. Metastasis is also angiogenesis-dependent: for a tumorcell to metastasize successfully, it generally gains access to thevasculature in the primary tumor, survive the circulation, arrest in themicrovasculature of the target organ, exit from this vasculature, growin the target organ, and induce angiogenesis at the target site. Thus,angiogenesis appears to be necessary at the beginning as well as thecompletion of the metastatic cascade.

The criticality of angiogenesis to the growth and metastasis ofneoplasms thus provides a target for therapeutic efforts. Appropriateanti-angiogenic agents may act directly or indirectly to influencetumor-associated angiogenesis either by delaying its onset or byblocking the sustainability of neovascularization of tumors.

Additional anti-cancer agents include biological agents (biotherapy),chemotherapy agents, and radiotherapy agents. More generally, theseother compositions would be provided in a combined amount effective tokill or inhibit proliferation of the cell.

Typically, vaccinia virus therapy will precede other agents by intervalsranging from days to weeks. In embodiments where the other agent andpoxvirus are applied separately to the cell, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agent and poxvirus would still be able toexert an advantageously combined effect on the cell. In such instances,it is contemplated that one may contact the cell with both modalitieswithin about 2-20 weeks of each other. In some situations, it may bedesirable to extend the time period for treatment significantly whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

In addition to treating a subject with vaccinia virus and ananti-angiogenic agent, an additional therapy can be used that includestraditional cancer therapies.

1. Chemotherapy

Cancer therapies include a variety of combination therapies with bothchemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, Temazolomide (an aqueous form of DTIC), or any analog orderivative variant of the foregoing. The combination of chemotherapywith biological therapy is known as biochemotherapy.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as .gamma.-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

3. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The combination of therapeuticmodalities, i.e., direct cytotoxic activity and inhibition or reductionof certain poxvirus polypeptides would provide therapeutic benefit inthe treatment of cancer.

4. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

5. Other Agents

Another form of therapy for use in conjunction with the current methodsincludes hyperthermia, which is a procedure in which a patient's tissueis exposed to high temperatures (up to 106° F.). External or internalheating devices may be involved in the application of local, regional,or whole-body hyperthermia. Local hyperthermia involves the applicationof heat to a small area, such as a tumor. Heat may be generatedexternally with high-frequency waves targeting a tumor from a deviceoutside the body. Internal heat may involve a sterile probe, includingthin, heated wires or hollow tubes filled with warm water, implantedmicrowave antennae, or radiofrequency electrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen.

B. Administration

In treating a tumor, the methods of the present invention administer anoncolytic vaccinia virus and then at a later time administer acomposition comprising an anti-angiogenic agent. The routes ofadministration will vary, naturally, with the location and nature of thetumor, and include, e.g., intradermal, transdermal, parenteral,intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., inthe proximity of a tumor, particularly with the vasculature or adjacentvasculature of a tumor), percutaneous, intratracheal, intraperitoneal,intraarterial, intravesical, intratumoral, inhalation, perfusion,lavage, and oral administration. Compositions will be formulatedrelative to the particular administration route.

Intratumoral injection, or injection directly into the tumor vasculatureis specifically contemplated. Local, regional or systemic administrationalso may be appropriate. For tumors of >4 cm, the volume to beadministered will be about 4-10 ml (preferably 10 ml), while for tumorsof <4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).Multiple injections delivered as single dose comprise about 0.1 to about0.5 ml volumes. The virus can be administered in multiple injections tothe tumor, spaced at approximately 1 cm intervals. In the case ofsurgical intervention, the present invention may be used preoperatively,to render an inoperable tumor subject to resection. Continuousadministration also may be applied where appropriate, for example, byimplanting a catheter into a tumor or into tumor vasculature. Suchcontinuous perfusion may take place for a period from about 1-2 hours,to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about1-2 days, to about 1-2 wk or longer following the initiation oftreatment. Generally, the dose of the therapeutic composition viacontinuous perfusion will be equivalent to that given by a single ormultiple injections, adjusted over a period of time during which theperfusion occurs. It is further contemplated that limb or organperfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of hepatic tumors,melanomas, and sarcomas.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Certain types of tumor will require more aggressive treatment, while atthe same time, certain patients cannot tolerate more taxing protocols.The clinician will be best suited to make such decisions based on theknown efficacy and toxicity (if any) of the therapeutic formulations.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently be described in terms of plaque forming units (pfu) fora viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹ 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher. Alternatively, depending onthe kind of virus and the titer attainable, one will deliver 1 to 100,10 to 50, 100-1000, or up to about or at least about 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, or1×10¹⁵ or higher infectious viral particles (vp), including all valuesand ranges there between, to the tumor or tumor site.

C. Injectable Compositions and Formulations

The preferred method for the delivery of an expression construct orvirus encoding all or part of a poxvirus genome to cancer or tumor cellsin the present invention is via intratumoral injection. However, thepharmaceutical compositions disclosed herein may alternatively beadministered parenterally, intravenously, intradermally,intramuscularly, transdermally or even intraperitoneally as described inU.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specificallyincorporated herein by reference in its entirety).

Injection of nucleic acid constructs may be delivered by syringe or anyother method used for injection of a solution, as long as the expressionconstruct can pass through the particular gauge of needle required forinjection. A novel needleless injection system has recently beendescribed (U.S. Pat. No. 5,846,233) having a nozzle defining an ampulechamber for holding the solution and an energy device for pushing thesolution out of the nozzle to the site of delivery. A syringe system hasalso been described for use in gene therapy that permits multipleinjections of predetermined quantities of a solution precisely at anydepth (U.S. Pat. No. 5,846,225).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral and intraperitonealadministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

II. Vaccinia Virus JX-594

Poxviruses have been known for centuries, with the characteristic pockmarks produced by variola virus (smallpox) giving this family its name.It appears that smallpox first emerged in China and the Far East over2000 years ago. Fortunately, this often fatal virus has now beeneradicated, with the last natural outbreak occurring in 1977 in Somalia.

The poxvirus viral particle is oval or brick-shaped, measuring some200-400 nm long. The external surface is ridged in parallel rows,sometimes arranged helically. The particles are extremely complex,containing over 100 distinct proteins. The extracellular forms containtwo membranes (EEV—extracellular enveloped virions), whereasintracellular particles only have an inner membrane (IMV—intracellularmature virions). The outer surface is composed of lipid and protein thatsurrounds the core, which is composed of a tightly compressednucleoprotein. Antigenically, poxviruses are also very complex, inducingboth specific and cross-reacting antibodies. There are at least tenenzymes present in the particle, mostly concerned with nucleic acidmetabolism/genome replication.

The genome of the poxvirus is linear double-stranded DNA of 130-300 Kbp.The ends of the genome have a terminal hairpin loop with several tandemrepeat sequences. Several poxvirus genomes have been sequenced, withmost of the essential genes being located in the central part of thegenome, while non-essential genes are located at the ends. There areabout 250 genes in the poxvirus genome.

Replication takes place in the cytoplasm, as the virus is sufficientlycomplex to have acquired all the functions necessary for genomereplication. There is some contribution by the cell, but the nature ofthis contribution is not clear. However, even though poxvirus geneexpression and genome replication occur in enucleated cells, maturationis blocked, indicating some role by the cell.

The receptors for poxviruses are not generally known, but probably aremultiple in number and on different cell types. For vaccinia, one of thelikely receptors is EGF receptor (McFadden, 2005). Penetration may alsoinvolve more than one mechanism. Uncoating occurs in two stages: (a)removal of the outer membrane as the particle enters the cell and in thecytoplasm, and (b) the particle is further uncoated and the core passesinto the cytoplasm.

Once into the cell cytoplasm, gene expression is carried out by viralenzymes associated with the core. Expression is divided into 2 phases:early genes: which represent about of 50% genome, and are expressedbefore genome replication, and late genes, which are expressed aftergenome replication. The temporal control of expression is provided bythe late promoters, which are dependent on DNA replication for activity.Genome replication is believed to involve self-priming, leading to theformation of high molecular weight concatamer, which are subsequentlycleaved and repaired to make virus genomes. Viral assembly occurs in thecytoskeleton and probably involves interactions with the cytoskeletalproteins (e.g., actin-binding proteins). Inclusions form in thecytoplasm that mature into virus particles. Cell to cell spread mayprovide an alternative mechanism for spread of infection. Overall,replication of this large, complex virus is rather quick, taking just 12hours on average.

At least nine different poxviruses cause disease in humans, but variolavirus and vaccinia are the best known. Variola strains are divided intovariola major (25-30% fatalities) and variola minor (same symptoms butless than 1% death rate). Infection with both viruses occurs naturallyby the respiratory route and is systemic, producing a variety ofsymptoms, but most notably with variola characteristic pustules andscarring of the skin.

A. Vaccinia Virus

Vaccinia virus is a large, complex enveloped virus having a lineardouble-stranded DNA genome of about 190K by and encoding forapproximately 250 genes. Vaccinia is well-known for its role as avaccine that eradicated smallpox. Post-eradication of smallpox,scientists have been exploring the use of vaccinia as a tool fordelivering genes into biological tissues (gene therapy and geneticengineering). Vaccinia virus is unique among DNA viruses as itreplicates only in the cytoplasm of the host cell. Therefore, the largegenome is required to code for various enzymes and proteins needed forviral DNA replication. During replication, vaccinia produces severalinfectious forms which differ in their outer membranes: theintracellular mature virion (IMV), the intracellular enveloped virion(IEV), the cell-associated enveloped virion (CEV) and the extracellularenveloped virion (EEV). IMV is the most abundant infectious form and isthought to be responsible for spread between hosts. On the other hand,the CEV is believed to play a role in cell-to-cell spread and the EEV isthought to be important for long range dissemination within the hostorganism.

Vaccinia virus is closely related to the virus that causes cowpox. Theprecise origin of vaccinia is unknown, but the most common view is thatvaccinia virus, cowpox virus, and variola virus (the causative agent forsmallpox) were all derived from a common ancestral virus. There is alsospeculation that vaccinia virus was originally isolated from horses. Avaccinia virus infection is mild and typically asymptomatic in healthyindividuals, but it may cause a mild rash and fever, with an extremelylow rate of fatality. An immune response generated against a vacciniavirus infection protects that person against a lethal smallpoxinfection. For this reason, vaccinia virus was used as a live-virusvaccine against smallpox. The vaccinia virus vaccine is safe because itdoes not contain the smallpox virus, but occasionally certaincomplications and/or vaccine adverse effects may arise, especially ifthe vaccine is immunocompromised.

As discussed above, vaccinia viruses have been engineered to express anumber of foreign proteins. One such protein is granulocyte-macrophagecolony stimulating factor, or GM-CSF. GM-CSF is a protein secreted bymacrophages that stimulates stem cells to produce granulocytes(neutrophils, eosinophils, and basophils) and macrophages. Human GM-CSFis glycosylated at amino acid residues 23 (leucine), 27 (asparagine),and 39 (glutamic acid) (see U.S. Pat. No. 5,073,627, incorporated byreference). GM-CSF is also known as molgramostim or, when the protein isexpressed in yeast cells, sargramostim (trademarked Leukine®), which isused as a medication to stimulate the production of white blood cells,especially granulocytes and macrophages, following chemotherapy. Avaccinia virus expressing GM-CSF has previously been reported. However,it was delivered not as an oncolytic agent, but merely as a deliveryvector for GM-CSF. As such, it has been administered to patients atdosage below that which can achieve significant oncolysis. Herein isdescribed the use of a GM-CSF expressing vaccinia virus that, in someembodiments, is administered at concentrations greater than 1×10⁸ pfu orparticles.

Vaccinia virus may be propagated using the methods described by Earl andMoss in Ausubel et al., 1994, which is incorporated by reference herein.

III. Nucleic Acid Compositions

In certain embodiments, the present invention concerns vaccinia virusand variants thereof.

A. Variants of Viral Polypeptides

Amino acid sequence variants of the polypeptides encoded by the vacciniavirus vectors of the invention can be substitutional, insertional ordeletion variants. A mutation in a gene encoding a viral polypeptide mayaffect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500 or more non-contiguous or contiguous amino acidsof the polypeptide, as compared to wild-type. Various polypeptidesencoded by Vaccinia virus may be identified by reference to Rosel etal., 1986, Goebel et al., 1990 and GenBank Accession Number NC001559,each of which is incorporated herein by reference.

Deletion variants lack one or more residues of the native or wild-typeprotein. Individual residues can be deleted or all or part of a domain(such as a catalytic or binding domain) can be deleted. A stop codon maybe introduced (by substitution or insertion) into an encoding nucleicacid sequence to generate a truncated protein. Insertional mutantstypically involve the addition of material at a non-terminal point inthe polypeptide. This may include the insertion of an immunoreactiveepitope or simply one or more residues. Terminal additions, calledfusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid (see Table 1, below).

TABLE 1 Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser SAGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val VGUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art byte and Kyte and Doolittle, 1982. It is acceptedthat the relative hydropathic character of the amino acid contributes tothe secondary structure of the resultant protein, which in turn definesthe interaction of the protein with other molecules, for example,enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

B. Polynucleotides Encoding Native Proteins or Modified Proteins

The present invention concerns polynucleotides, isolatable from cells,that are capable of expressing all or part of a protein or polypeptide.In some embodiments of the invention, it concerns a viral genome thathas been specifically mutated to generate a virus that lacks certainfunctional viral polypeptides. The polynucleotides may encode a peptideor polypeptide containing all or part of a viral amino acid sequence orthey be engineered so they do not encode such a viral polypeptide orencode a viral polypeptide having at least one function or activityreduced, diminished, or absent. Recombinant proteins can be purifiedfrom expressing cells to yield active proteins. The genome, as well asthe definition of the coding regions of Vaccinia Virus may be found inRosel et al., 1986; Goebel et al., 1990; and/or GenBank Accession NumberNC_(—)001559, each of which is incorporated herein by reference.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a polypeptide refers to a DNA segmentthat contains wild-type, polymorphic, or mutant polypeptide-codingsequences yet is isolated away from, or purified free from, totalmammalian or human genomic DNA. Included within the term “DNA segment”are a polypeptide or polypeptides, DNA segments smaller than apolypeptide, and recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like.

As used in this application, the term “poxvirus polynucleotide” refersto a nucleic acid molecule encoding a poxvirus polypeptide that has beenisolated free of total genomic nucleic acid. Similarly, a “vacciniavirus polynucleotide” refers to a nucleic acid molecule encoding avaccinia virus polypeptide that has been isolated free of total genomicnucleic acid. A “poxvirus genome” or a “vaccinia virus genome” refers toa nucleic acid molecule that can be provided to a host cell to yield aviral particle, in the presence or absence of a helper virus. The genomemay or may have not been recombinantly mutated as compared to wild-typevirus.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a particular polypeptide from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 above).

Similarly, a polynucleotide comprising an isolated or purified wild-typeor mutant polypeptide gene refers to a DNA segment including wild-typeor mutant polypeptide coding sequences and, in certain aspects,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein,polypeptide, or peptide-encoding unit (including any sequences requiredfor proper transcription, post-translational modification, orlocalization). As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences, and smallerengineered gene segments that express, or may be adapted to express,proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

A nucleic acid encoding all or part of a native or modified polypeptidemay contain a contiguous nucleic acid sequence encoding all or a portionof such a polypeptide of the following lengths: 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480,490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000,or more nucleotides, nucleosides, or base pairs.

The term “recombinant” may be used in conjunction with a polypeptide orthe name of a specific polypeptide, and this generally refers to apolypeptide produced from a nucleic acid molecule that has beenmanipulated in vitro or that is the replicated product of such amolecule.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the presentinvention may encode full-length polypeptide from any source or encode atruncated version of the polypeptide, for example a truncated vacciniavirus polypeptide, such that the transcript of the coding regionrepresents the truncated version. The truncated transcript may then betranslated into a truncated protein. A tag or other heterologouspolypeptide may be added to the modified polypeptide-encoding sequence,wherein “heterologous” refers to a polypeptide that is not the same asthe modified polypeptide.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence acontiguous nucleic acid sequence from that shown in sequences identifiedherein (and/or incorporated by reference). Such sequences, however, maybe mutated to yield a protein product whose activity is altered withrespect to wild-type.

It also will be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of these identifiedsequences. Recombinant vectors and isolated DNA segments may thereforevariously include the poxvirus-coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includepoxvirus-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent poxvirus proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencythat are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein.

C. Mutagenesis of Poxvirus Polynucleotides

In various embodiments, the poxvirus polynucleotide may be altered ormutagenized. Alterations or mutations may include insertions, deletions,point mutations, inversions, and the like and may result in themodulation, activation and/or inactivation of certain pathways ormolecular mechanisms or particular proteins (e.g., thymidine kinase), aswell as altering the function, location, or expression of a geneproduct, in particular rendering a gene product non-functional. Whereemployed, mutagenesis of a polynucleotide encoding all or part of aPoxvirus may be accomplished by a variety of standard, mutagenicprocedures (Sambrook et al., 1989).

Mutations may be induced following exposure to chemical or physicalmutagens. Such mutation-inducing agents include ionizing radiation,ultraviolet light and a diverse array of chemical such as alkylatingagents and polycyclic aromatic hydrocarbons all of which are capable ofinteracting either directly or indirectly (generally following somemetabolic biotransformations) with nucleic acids. The DNA damage inducedby such agents may lead to modifications of base sequence when theaffected DNA is replicated or repaired and thus to a mutation. Mutationalso can be site-directed through the use of particular targetingmethods.

D. Vectors

To generate mutations in the poxvirus genome, native and modifiedpolypeptides may be encoded by a nucleic acid molecule comprised in avector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which an exogenous nucleic acid sequence can be insertedfor introduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al., (1989)and Ausbel et al., 1994, both incorporated herein by reference. Inaddition to encoding a modified polypeptide such as modified gelonin, avector may encode non-modified polypeptide sequences such as a tag ortargeting molecule.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. Nos. 4,683,202, 5,928,906, eachincorporated herein by reference). Furthermore, it is contemplated thecontrol sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (1989), incorporated herein by reference. The promoters employedmay be constitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse α2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996), and the SM22α promoter.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′-methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading flames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (NCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, incorporated herein by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “on”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

E. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses (which does notqualify as a vector if it expresses no exogenous polypeptides). A hostcell may be “transfected” or “transformed,” which refers to a process bywhich exogenous nucleic acid, such as a modified protein-encodingsequence, is transferred or introduced into the host cell. A transformedcell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingyeast cells, insect cells, and mammalian cells, depending upon whetherthe desired result is replication of the vector or expression of part orall of the vector-encoded nucleic acid sequences. Numerous cell linesand cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla, Calif.). Alternatively, bacterial cells such asE. coli LE392 could be used as host cells for phage viruses. Appropriateyeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, andPichia pastoris.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

F. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnon-viral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

G. Lipid Components and Moieties

In certain embodiments, the present invention concerns compositionscomprising one or more lipids associated with a nucleic acid, an aminoacid molecule, such as a peptide, or another small molecule compound. Inany of the embodiments discussed herein, the molecule may be either apoxvirus polypeptide or a poxvirus polypeptide modulator, for example anucleic acid encoding all or part of either a poxvirus polypeptide, oralternatively, an amino acid molecule encoding all or part of poxviruspolypeptide modulator. A lipid is a substance that is characteristicallyinsoluble in water and extractable with an organic solvent. Compoundsthan those specifically described herein are understood by one of skillin the art as lipids, and are encompassed by the compositions andmethods of the present invention. A lipid component and a non-lipid maybe attached to one another, either covalently or non-covalently.

A lipid may be naturally-occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

A nucleic acid molecule or amino acid molecule, such as a peptide,associated with a lipid may be dispersed in a solution containing alipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid or otherwise associated with a lipid. A lipid orlipid/poxvirus-associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine(Gibco BRL)-poxvirus orSuperfect (Qiagen)-poxvirus complex is also contemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

IV. Examples

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Preclinical studies were performed in murine HCC models to assessmechanisms of JX-594-induced vascular shutdown and subsequentre-perfusion. The potential of sorafenib to block reperfusion wasevaluated. In a Phase 2 clinical trial, patients with HCC were treatedwith JX-594 by intratumoral injection every two weeks for three cycles.Tumor size, blood flow and density were assessed by dynamiccontrast-enhanced (dce)-MRI and DW-MRI at baseline, Day 5 and Week 8.Two patients with partial tumor re-perfusion at Week 8 initiatedstandard sorafenib therapy, and sequential DCE-MRI scans were performed.

Tumor vascular shutdown was demonstrated in a murine HCC model after ITor IV administration of JX-594. Vascular shutdown was dependent on viralreplication; mGM-CSF expression from the virus and neutrophilinfiltration enhanced the effect. Re-perfusion of the tumor rim wasdemonstrated over time and correlated to increased VEGF levels in thetumor. Adjuvant sorafenib therapy inhibited angiogenesis and led tosignificantly improved anti-tumoral efficacy over either agent alone.HCC patients treated with JX-594 demonstrated acute tumor vascularshutdown and necrosis in both injected and non-injected tumors withinthe liver. Adjuvant therapy with sorafenib after the Week 8 assessmentled to dramatic and durable tumor necrosis and vascular shutdown in twopatients. Reviews of serial MRI scans from HCC patients on sorafenibalone demonstrated that these findings were specific to patientspre-treated with JX-594.

Because tumors have a high mutation rate and therefore may have a highdegree of heterogeneity leading to mixed responses to any one therapy,it can be useful to combine certain anti-cancer agents with alternativemechanisms of action. To determine if the combination of oncolytic virustherapy with sorafenib was safe and effective, in vitro and in vivopreclinical experiments were conducted.

Example 1 In Vitro Data—Sorafenib Inhibits JX-594

JX-594 was tested in combination with sorafenib on the human HCC cellline PLC/PRF/5. The PLC/PRF/5 human HCC cell line supports JX-594replication. However, when Sorafenib (10 μM) was added either 2 hrsprior to; during; or 2 hrs after JX-594 infection, burst size wasdecreased up to 100 fold (FIG. 1). JX-594 was also tested in combinationwith sorafenib on human ovarian cancer cell line A2780 and human HCCline HepG2.

Cell Culture and Sorafenib Preparation: Human tumor cell lines A2780(ovarian) and HepG2 (HCC) were obtained from American Type CultureCollection (ATCC). Cells were cultured in DMEM supplemented with 10% FBSand 1% pen/strep. Cells were grown at 37° C. in a humidified incubatorcontaining 5% CO₂. For in vitro use, sorafenib was dissolved in DMSO(Sigma-Aldrich Corp.) to a concentration of 1 mg/ml and further dilutedto appropriate final concentration (100 ng/ml, 250 ng/ml, 500 ng/ml,1000 ng/ml and 2500 ng/ml) in DMEM with 10% fetal bovine serum. DMSO inthe final solution did not exceed 0.2% (v/v).

Plaque Formation, Burst Assay and Cell Viability: A2780 or HepG2 cellswere seeded into 6-well plates at 4×10⁵ cells/well and left overnight.100 pfu of JX-594 was then added to each well and allowed to infect for2 hours. At the end of the infection, the media was removed and 3%Carboxymethylcellulose DMEM overlay containing sorafenib at finalconcentrations of 0, 0.1, 0.25, 0.5, 1.0, and 2.5 μg/mL was added. Threedays later, plates were stained with crystal violet and plaques werecounted. In parallel, to assess replication (burst size), 6-well plateswere prepared as above. Instead of staining and counting plaques, cellswere harvested from each well for purification of virus. Cells werelysed by 3 rounds of freezing and thawing followed by sonication, beforeserial dilutions of the crude viral lysate was added to A2780 cells totiter the virus by plaque assay. Furthermore, to assess the directeffects of sorafenib on cell viability, cells were plated in 96 wellplates and incubated with sorafenib only. Cell viability was determinedby means of colorimetric assay based on live-cell mediated reduction oftetrazolium salt to formazan chromagen (Cell Counting Kit-8, DonjindoLaboratories, Kumamoto, Japan).

Results: Concentrations of Sorafenib that are below cytotoxic levels caninhibit viral growth and replication on A2780 and HEPG2 tumor cell lines(FIG. 2). Additional experiments showed that sorafenib inhibited JX-594replication in other human HCC lines including SNU423, SNU398, SNU475,SNU449, SNU387 and the human osteosarcoma line U2OS.

Sorafenib is a multikinase inhibitor and is capable of inhibiting theRas signal transduction pathway (RAS/RAF/MEK/ERK) by inhibition ofintracellular serine/threonine kinases Raf-1 and B-Raf. Activation ofthe Ras/Raf pathway commonly occurs in cancer and leads totranscriptional activation of E2F-responsive genes, including S-Phasegenes such as thymidine kinase (TK) (Hengstschlager et al., 1994).JX-594 is vaccinia virus attenuated by inactivation of the viral TKgene. This mutation is designed to provide for selective replication incells with high levels of cellular TK (i.e. cancer cells with abnormallyactivated pathways leading to constitutive E2F activation). Whilesorafenib and JX-594 are therefore both designed to targeted cancercells with activated Ras pathway, simultaneous dosing of the therapiesin vitro can lead to inhibition of viral replication which depends on anactivated Ras pathway.

Example 2 In Vivo Data—Sorafenib Enhances JX-594

Contrary to the in vitro findings, preclinical efficacy models showedthat combining sorafenib and JX-594 shows better efficacy than eitheragent alone.

Sorafenib enhances JX-594 activity against murine CT26 primary tumors invivo: The combination of JX-594 and sorafenib was tested in animmunocompentent murine model of subcutaneously implanted CT26colorectal tumor cells. Balb/c mice were injected with 3×10⁵ CT26 cellssubcutaneously and after 11 days, treatment commenced with Sorafenibalone (10 mg/kg p.o. daily for 2 weeks), JX594 alone (10⁸ pfu IV 3 timesper week for 1 week), or in combination (JX594 prior to Sorafenib, orSorafenib prior to JX594) (n=4 per group) (FIG. 3, upper panel). Controlanimals received PBS only. For this experiment, a version of JX-594 wasused that expresses murine GM-CSF. Tumors were measured twice weeklyuntil endpoint. Surviving proportions of each study group at eachtimepoint post treatment are shown (FIG. 3, middle panel). The meantumor volumes of each study group at each timepoint post-treatment areshown (FIG. 3, lower panel). Compared to JX-594 alone, JX-594 incombination with Sorafenib enhanced survival and reduced tumor burden.JX-594 in combination with Sorafenib also reduced the tumor burdencompared to Sorafenib-only animals. The preferred regimen was JX-594followed by Sorafenib.

Sorafenib Enhances JX-594 Activity Against Murine B16 Melanoma LungNodules in Vivo: The combination of JX-594 and sorafenib was tested inan immunocompentent murine model of B16 melanoma tumors. C57BL/6 micewere injected with 3×10⁵ B16-F10-LacZ cells IV and treated withSorafenib alone (HD: 10 mg/kg, LD: 50 μg/kg p.o. daily for 2 weeks),JX594 alone (10⁷ pfu IV 3 times per week for 1 week), or in combination(LD or HD sorafenib prior to JX-594 IV) (n=5 per group)(FIG. 4, toppanel). At the end of treatment, mice were sacrificed and lungs werefixed and stained to detect B16 surface nodules (n=5 per group). Themean number of tumor nodules at the end of study is shown for each group(FIG. 4). In the 50 μg/kg p.o. Sorafenib +JX-594 group, there wassignificant reduction in B16 tumors compared to the JX-594 alone groupor the Sorafenib alone group.

Sorafenib Enhances JX-594 Activity Against Human HCC Xenograft Model inVivo: SCID mice were implanted with subcutaneous HepG2 humanhepatocellular carcinoma xenograft tumors. Once tumors reached a size ofapproximately 12-14 mm maximal diameter mice were randomized into one ofsix treatment groups (n=8 per group) (1) PBS alone, (2) sorafenib alone(standard daily intraperitoneal dosing with 400 μg), (3) JX-594 alone(intravenous treatment of 10⁷ pfu weekly for six total doses), (4)simultaneous treatment with JX-594 and sorafenib, (5) sorafenib followedby JX-594 and (6) JX-594 (2 doses) followed by sorafenib.

Tumor size was measured using calipers and mice euthanized when tumorburdens reached allowable limit for ethical purposes. The regimen ofJX-594 followed by sorafenib was superior to control or either agentalone in terms of tumor growth and time-to-tumor progression. Inaddition, this sequence was superior to sorafenib followed by JX-594 andto simultaneous treatment (FIG. 5).

A. Preclinical HCC Tumor Model Studies of Vascular Shutdown

Experimental Methods, Pilot Experiment: The pilot experiment wasdesigned to show that JX-594 followed by Sorafenib induces acutevascular shutdown in the HepG2 model. Three groups were used (n=5animals each). Group 1 received PBS only; Group 2 received a single doseof 10⁶ pfu JX-594 administered intravenously (IV); and Group 3 receiveda single dose of 10⁶ pfu JX-594 administered intratumorally (IT). Serumsamples were collected on D5 for measurement of VEGF and GM-CSF levels.Prior to euthanization, mice were injected with fluorescent microspheresto allow for visualization of tumor perfusion. The resected tumor wasdivided into three portions for analysis 1) Flash frozen sample formeasurement of VEGF protein levels 2) OCT-embedded sample preparation offrozen tumor sections to visualize microspheres and vascular marker CD31and 3) Formalin fixed/paraffin-embedded sample for histologicalanalysis.

Experimental Methods, Vascularity Study: The study followed longer termeffects on vascularity and assessed the efficacy of JX-594+/−Sorafenibin the HepG2 model. HepG2 (human HCC line) tumors were implantedsubcutaneously in nude mice. Animals were randomized to seven treatmentgroups (n=5 per group) to test agents alone, and in combination. Groupswere treated with JX-594 (10⁶ pfu intratumorally, D1 and D8) and/orSorafenib (400 μg i.p., daily D15-D29) or PBS (control) on the scheduledetailed in (FIG. 6). Tumors were measured using calipers twice weekly.Serum samples were collected on D1, D8, D15, D22 and D29 for measurementof VEGF and GM-CSF levels. Mice were euthanized on D22 or D35. Prior toeuthanization, mice were injected with fluorescent microspheres to allowfor visualization of tumor perfusion. The resected tumor was dividedinto three portions for analysis 1) Flash frozen sample for measurementof VEGF protein levels 2) OCT-embedded sample preparation of frozentumor sections to visualize microspheres and vascular marker CD31 and 3)Formalin fixed/paraffin-embedded sample for histological analysis.

Results: Vessels were counted in CD31-stained OCT tumor sections; theaverage number of vessels in 3 fields of 200× magnification arepresented in (FIG. 6). Sorafenib alone caused a transient decrease inthe number of vessels (Day 29), but vessel counts increase again overtime (Day 35). However, in animals that received JX-594 followed bysorafenib, the number of vessel on D35 were lower compared to thesorafenib only group, suggesting the combination has a more lastingeffect on tumor vasculature.

B. Clinical Data

JX-594 is a first-in-class targeted oncolytic poxvirus designed toselectively replicate in and destroy cancer cells. Direct oncolysis plusgranulocyte macrophage—colony stimulating factor (GM-CSF) expressionalso stimulates tumor vascular shutdown in animal tumor models. Tumorvascular shutdown was assessed following JX-594 therapy in patients withhepatocellular carcinoma. In addition, feasibility of adjuvantanti-angiogenic therapy with sorafenib to prevent re-perfusion followingJX-594 in both preclinical and clinical studies of hepatocellularcarcinoma (HCC) were studied.

Methods: Preclinical studies were performed in murine HCC models toassess mechanisms of JX-594-induced vascular shutdown and subsequentre-perfusion. The potential of sorafenib to block reperfusion wasevaluated. In a Phase 2 clinical trial, patients with HCC were treatedwith JX-594 by intratumoral injection every two weeks for three cycles.Tumor size, blood flow and density were assessed by dynamiccontrast-enhanced (DCE)-MRI and DW-MRI at baseline, Day 5 and Week 8.Five patients with partial tumor re-perfusion at Week 8 initiatedstandard sorafenib therapy, and sequential dce-MRI scans were performed.

Findings: Tumor vascular shutdown occurs in murine HCC model after IT orIV administration of JX-594. Vascular shutdown was dependent on viralreplication; mGM-CSF expression from the virus and neutrophilinfiltration enhanced the effect. Re-perfusion of the tumor rim isdemonstrated over time and correlated with increased VEGF levels in thetumor. Adjuvant sorafenib therapy inhibited angiogenesis and led tosignificantly improved anti-tumoral efficacy over either agent alone.HCC patients treated with JX-594 demonstrated acute tumor vascularshutdown and necrosis in both injected and non-injected tumors withinthe liver. Adjuvant therapy with sorafenib after the Week 8 assessmentled to dramatic and durable tumor necrosis and vascular shutdown in atleast three patients. Reviews of serial MRI scans from 15 HCC patientson sorafenib alone demonstrated that these findings were enhanced andenriched in patients pre-treated with JX-594.

JX-594 causes acute vascular shutdown and necrosis in HCC tumors, andsensitizes HCC to subsequent therapy with sorafenib. Randomizedcontrolled trials of JX-594 followed by sorafenib versus sorafenib aloneare indicated. Sequential therapy regimens with oncolytic poxvirusesfollowed by anti-angiogenic agents hold promise.

Introduction: The targeted oncolytic poxvirus JX-594 replicatesselectively in cancer cells, resulting in virus progeny production,tumor cell necrosis, release and spread within tumor tissues. JX-594 isalso engineered to express the GM-CSF transgene in order to enhance theanti-tumoral immunity that results from oncolysis. The vaccinia backboneis inherently tumor-selective due to EGFR-ras pathway dependency andtumor-resistance to interferons. The inherent therapeutic index isamplified by the TK deletion; JX-594 replication is dependent oncellular TK, which is driven to high levels by cell cycle abnormalitiesin cancer. Results from a Phase 1 clinical trial of JX-594 in patientswith refractory liver tumors demonstrated safety, efficacy andmechanistic proof-of-concept for JX-594 replication, systemicdissemination and biologically-active GM-CSF expression. Recentlypublished preclinical studies demonstrated that oncolytic virus therapycan also induce acute tumor vascular shutdown (Breitbach, et al. 2007).

The inventors contemplate that the oncolytic poxvirus JX-594 causestumor vascular shutdown, and that the GM-CSF expression from the virusenhances neutrophil recruitment and activation leading to augmentationof anti-vascular effects. Following preclinical studies, the inventorsassess tumor vascular shutdown in patients with HCC, a tumor type thatis hypervascular. Preclinical and clinical studies demonstrated thepossibility of subsequent progression of a vascularized tumor rim.

Sorafenib is an oral multikinase inhibitor approved for treatment ofrenal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Sorafenibinhibits surface tyrosine kinase receptors (VEGF-R, PDGF-R) andintracellular serine/threonine kinases (Raf-1, B-Raf) and therefore is amultimechanistic anti-cancer agent. Sorafenib may affect tumor cellsdirectly by inhibiting the Ras signaling pathway (RAS/RAF/MEK/ERK) whichis commonly activated in cancer cells and promotes cell proliferation.Sorafenib may also reduce tumor growth through its anti-angiogeniceffects resulting from inhibition of VEGF-R. Sutent (sunitinib/SU11248)is another targeted cancer therapy that inhibits the actions of vascularendothelial growth factor (VEGF) and has anti-angiogenic effects. It isapproved for treatment of renal cell carcinoma and gastrointestinalstromal tumor (GIST).

The inventors contemplate that post-JX-594 re-perfusion is blocked bythe anti-angiogenic agent sorafenib which is approved for use in HCCpatients, or sutent which is approved for use in RCC patients. We testedthis hypothesis in preclinical models of HCC, and in five patients aftercompletion of therapy with JX-594.

C. Materials and Methods

Study Approvals and Registration: Study protocol and informed consentforms were approved by the US FDA, Korean FDA, and Institutional Reviewand Infection Control Committees at Pusan National University Hospital,Busan, South Korea. The protocol was registered via the world wide webat clinicaltrials.gov.

Patient Selection: Patients signed informed consent, according to GoodClinical Practice (GCP) guidelines. Inclusion criteria includedunresectable, injectable hepatocellular tumor(s) within the liver(primary HCC) that had progressed despite treatment with standardtherapies (treatment-refractory), normal hematopoietic function(leukocyte count >3, ×10⁹ cells/L, hemoglobin >100 g/L, platelet count>60×10⁹ cells/L) and organ function (including creatinine ≦132.6 μmol/L,aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ≦2·5 ofupper normal limit, Child-Pugh class A or B), life expectancy ≧16 weeks,and Karnofsky Performance Status (KPS)≧70. Exclusion criteria includedextrahepatic tumors, tumors >10 cm max diameter, increased risk forvaccination complications (e.g. immunosuppression, eczema), treatmentwith immunosuppressive or cancer treatment agents within 4 weeks,rapidly progressive ascites, pregnancy or nursing.

Manufacturing and Preparation of JX-594: JX-594 is a Wyeth strainvaccinia modified by insertion of the human CSF2 and LacZ genes into theTK gene region under control of the synthetic early-late promoter andp7.5 promoter, respectively. Clinical trial material (CTM) was generatedaccording to Good Manufacturing Practice (GMP) guidelines in Vero cellsand purified through sucrose gradient centrifugation. The genome-to-pfuratio was approximately 70:1. JX-594 was formulated inphosphate-buffered saline with 10% glycerol, 138 mM sodium chloride atpH 7.4. Final product quality control release tests included assays forsterility, endotoxin and potency. CTM was also tested for GM-CSF proteinconcentration and was negative (lower limit of detection <14,000 pg/mL).JX-594 was diluted in 0.9% normal saline in a volume equivalent to 25%of the estimated total volume of target tumor(s).

JX-594 Treatment Procedure, All IT Patients except 11301 (SutentPatient): Patients with unresectable HCC were randomized to receive oneof two dose levels (10⁸ or 10⁹ pfu). JX-594 was administered viaimaging-guided intratumoral injection using a multi-pronged Quadrafuseinjection needle in roughly spherical tumors, and by a 21-gauge PEIT(percutaneous ethanol injection, multi-pore; HAKKO Medicals; Tokyo,Japan) needle in irregularly-shaped tumors. Tumors (n=1-5) were injectedevery two weeks for three cycles. The same tumors injected on cycle 1were injected thereafter on each cycle.

Sorafenib Therapy and Tumor Response Assessment Following JX-594:Patients completed the IT clinical trial of JX-594 after 8 weeks onstudy. Some patients (Patients 1702, 1705, 1002, 1712, 1713) went on toreceive standard sorafenib treatment (400 mg twice daily p.o.). Tumorsin these patients were followed by DCE-MRI imaging using the sameprocedures that were used to assess response to JX-594 treatment.

JX-594 Treatment Procedure, Patients Treated by IV JX-594 Followed by ITJX-594 Followed by Sorafenib: Patients with unresectable HCC receivedtwo doses of JX-594 levels (10⁹ pfu). For the first dose (Day 1), JX-594was administred by intravenous infusion over 60 minutes. For the secondand third doses (Day 8 and Day 22), JX-594 was administered viaimaging-guided intratumoral injection using a multi-pronged Quadrafuseinjection needle in roughly spherical tumors, or by a 21-gauge PEIT(percutaneous ethanol injection, multi-pore; HAKKO Medicals; Tokyo,Japan) needle in irregularly-shaped tumors. Starting on Day 25, patientsimitated oral sorafenib therapy (400 mg twice daily p.o.). Patients withviable tumor tissue received a Week 12 IT injection of JX-594 (sorafenibtreatment was temporarily discontinued 2-3 days before, during and 4-5days after this booster injection). Imaging (CT, DECE MIR and/or PET-CT)was performed at baseline, Day 25, Week 6 and/or Week 12 to assessresponse.

JX-594 Treatment, Patient 11301 (Sutent Pateint): Patient 11301 hadrenal cell carcinoma that had metastasized to the liver. Liver tumorswere treated by intratumoral injection using a 21-gauge PEIT needle. Thepatient received a total of 4 doses of JX-594 (10⁹ pfu/dose) given threeweeks apart (=4 cycles). After every two cycles of treatment,contrast-enhanced CT scanning was performed and week 6 responseassessment was performed using RECIST and Choi criteria. Patientexperienced stable disease (SD) by RECIST critera and a Choi response(42% decrease in HU) (Park et al., 2008).

Sutent Treatment: Subsequently, Patient 11301 progressed and went on toreceive sutent treatment. Patient received 3 courses of 50 mg/daily (4weeks on, 2 weeks off) then 3 courses of 37.5 mg/daily (4 weeks on, 2weeks off), and was maintained on a schedule of 25 mg/daily (2 weeks on,1-2 weeks off).

Tumor Vascularity and Response Assessment: DCE MRI (dynamiccontrast-enhanced magnetic resonance imaging) was performed at screening(baseline), on Day 5 (optional) and Week 8. For patients going on toSorafenib, DCE MRI was performed 4 and/or 8 weeks after the start ofSorafenib treatment. DCE MRI assesses tumor size, vascularity andnecrosis. The screening/baseline DCE MRI was used as the reference fromwhich to determine time to progression and response rates. The Day 5(optional) DCE MRI was used to assess early effects such as acutevascular shutdown. Tumor progression status and tumor response(s) toJX-594 were assessed radiologically by modified RECIST and modified Choicriteria at the Week 8 visit. Independent review of the images was madeby radiologist(s) with expertise in evaluating hepatocellular carcinomaon MRI scans. For evaluation of the intra-hepatic tumors, the proportionof subjects with an objective “complete” or “partial” anti-tumorresponse was determined based on modified RECIST, and a response asmeasured by Modified Choi criteria (defined as a ≧10% decrease in thesum of the longest diameter and/or ≧15% decrease in the average tumorsignal intensity at MRI).

Tumor Response by Modified RECIST: The modifications to RECIST formeasurement of tumor response and tumor progression were as follows. Newtumor(s) that developed within the liver during or after treatment weremeasured. Their maximum diameter(s) was included in the sum of themaximum diameter of all intra-hepatic tumors. However, new tumors withinthe liver were not considered evidence for progression per se. Therationale for this RECIST criteria modification is the following. JX-594infection of a tumor mass that was originally undetectableradiographically may make that tumor appear to be new and/or progressivedue to inflammation and/or necrosis; however, these changes do notrepresent true tumor progression. In addition, because the treatmentgoal was to control the intra-hepatic tumor burden, new tumors detectedextra-hepatically in the abdomen were be noted (and recorded bylocation) but were not included in the determination of overallresponse. Thus tumor response or progression was determined by the sumof the longest diameters of measurable intra-hepatic tumors anddetermined as follows: Complete Response (CR): Disappearance of alltumor(s). Partial Response (PR): At least a 30% decrease in the sum ofthe LD of tumor(s), taking as reference the baseline sum LD. ProgressiveDisease (PD): At least a 20% increase in the sum of the LD of tumor(s),taking as reference the baseline sum LD. Stable Disease (SD): Neithersufficient shrinkage to qualify for PR nor sufficient increase toqualify for PD, taking as reference the baseline sum LD.

Tumor Response by Modified Choi Criteria: The Choi response criteriatakes into consideration changes in tumor density in addition to tumordiameter (versus RECIST) and data supporting its utility has beenpublished (Choi et al. 2004). Early studies have shown that liver tumorstreated with JX-594 can develop significant internal necrosis without aconcomitant decrease in size, as has been described in gastrointestinalstromal tumors by Choi et al. (2004). Therefore, intra-hepatic tumorswere also measured and response evaluated using a Modified ChoiCriteria. The modification of the criteria is necessary as DCE MRI wasthe imaging modality employed for measuring tumor response. As MRI doesnot have a standardized system of contrast density measurements similarto CTs Hounsfield units, a comparison of percent enhancement of tumorsat baseline and following treatment was performed using region ofinterest (ROI) signal intensity (SI) measurements. Thescreening/baseline DCE MRI was used as the reference from which todetermine response. A response by Modified Choi criteria was defined asa ≧10% decrease in the sum of the longest diameter of the injectedtumor(s) and/or ≧15% decrease in the average injected tumor signalintensity on MRI. The average MRI signal intensity (SI) was measured asa percentage of tumor.

MRI Imaging Protocols: Each patient will undergo MRI of the abdomen,including dynamic contrast-enhanced magnetic resonance imaging (DCEMRI), at time points prescribed by the protocol. Imaging will beperformed on a 1.5 T or 3.0 T MR system using a body/torso array coilpositioned for complete imaging coverage of the liver with the patientin the supine position. A dielectric pad may be placed over the liver.

Typically imaging parameters are fixed between patient follow-up visits.While the sequences below list a range of acceptable parameters, once apatient has had their initial scan, these parameters should be employedon all subsequent scans. In addition, a patient should be scanned usingthe same MRI scanner.

An intravenous line will be started prior to the examination with thelargest gauge catheter possible placed in a peripheral vein with normalsaline running at KVO. Alternatively, for patients with PICC lines orexternal venous catheter ports which are compatible with automatedcontrast injectors, these may be used for venous access. Extracellulargadolinium chelate contrast will be administered by intravenous bolusinjection at 0.1 mmol/kg dose and at a rate of 2 cc/second via anautomated injector, followed by an immediate injection of 20 cc saline.Any variations from the injection rate or dose, or extravasation ofcontrast, will be noted in the CRF.

Imaging Protocol for 1.5 Tesla MR Systems: The following pulse sequenceswill be performed:

Precontrast Imaging: (1) 2D Axial In- and Opposed Phase T1: T1-weightedspoiled gradient echo (SPGR) dual phase axial images (TR: shortestpossible; TE: 2.1 and 4.2; flip angle (FA): 80-90 degrees, slicethickness 5-7 mm, slice gap maximum 1 mm; phase encodes 160-192interpolated to 256×256; field of view optimized to the patient's bodyhabitus, 300-450 mm. (2) 2D FSE T2 Axial: TR: 3500-5000 msec(effective); TE: 60-88 msec; phase encodes: 160−256×256; field of viewoptimized to the patient's body habitus, 300 150 mm; slice thickness,5-7 mm; maximum slice gap 1 mm; imaging should be performed with fatsuppression. Respiratory trigging or other motion suppression techniquesare encouraged.

DCE-MRI: (3) 3D T1 Dynamic imaging: A total of 6 sets of this sequenceare performed: one precontrast, 4 immediate postcontrast, and one 5minute delayed image set. Parameters for the 3D T1-weightedfat-suppressed acquisitions are as follows: TR=2.0-4.5 msec; TE=1.42-2.0msec; flip angle, 8-12°; phase encodes 160-192 interpolated to 512×512;field of view optimized to the patient's body habitus, 300-450 mm;interpolated section thickness, 1.5-3 mm; slab thickness to ensurecomplete coverage of the liver.

To determine the timing for the first contrast-enhanced acquisition(hepatic arterial phase), a 1-2 mL test bolus of contrast material willbe administered and the circulation time (time to peak arterialenhancement) will be set as the acquisition delay time. Alternatively,if automated timing software is available to determine arterial phaseenhancement, this may also be used. The 4 postcontrast dynamic sequenceswill be performed with a 40 second time gap between each acquisition. Anadditional delayed sequence will also be acquired at 5 minutes followinginjection (for a total of 5 post-contrast sequences). All acquisitionswill be performed during suspended respiration, either inspiration orexpiration based on institutional practices. The system does not undergoany tuning changes between the pre- and post-contrast sequences. Anyvariations from this imaging protocol will be noted in the CRF.

Imaging Protocol for 3.0 Tesla MR Systems: The following pulse sequenceswill be performed:

Precontrast Imaging: (1) T1w 2D Axial In-Phase (IP) and Out-of-Phase(OP): dual-phase spoiled gradient echo (SPGR). FOV: optimized to bodyhabitus, 300-450 mm; TR: minimum to cover liver; TE: default in phaseand opposed phase TE's; Flip angle: 80-90 degrees; Slice thickness: 5-7mm; Gap: 0-1 mm (O-mm gap preferred); Frequency matrix: 320; Phaseencodes: 160-224, interpolated to 512×512; Fat sat: off; Bandwidth:default setting.

(2) T2w 2D Axial SSFSE. FOV: use same as (1) above; TR: shortesteffective TR to image complete liver; TE effective: 60-88 msec; Slicethickness: use same as (1); Gap: use same (1); Frequency matrix: 320;Phase encodes: 160-224, interpolated to 512×512; Fat sat: off.

(3) T2w 2D Axial FSE. Either free breathing with respiratory triggeringor breathhold imaging can be used here. However, it will be standardizedwithin a patient's exams. For example, if a patient is scanned atbaseline using respiratory triggering, all subsequent MR exams will userespiratory triggering with this sequence. If respiratory triggering isused, an echo train length of 12-20 should be employed, as should besufficient excitations/acquisitions for optimal signal to noise. Ifbreath-hold T2 imaging is performed, employ an echo train length of24-32 and 1 acquisition. FOV: use same as (1) above. TR effective:3500-5000; Slice thickness: use same as (1); Gap: use same as (1);Frequency matrix: 320; Phase encodes: 160-224, interpolated to 512×512;Fat sat: on. DCE MRI (4) T1 w 3D Axial spoiled gradient echo (SPGR).FOV: use same as (1) above; TR: 2-5 msec; TE: 1.4-2.5; Flip angle: 8-15;Slice thickness: 1.5-3 mm interpolated; Slab thickness: cover entireliver; Frequency matrix: 288-320; Phase encodes: 160-224, interpolatedto 512×512; Fat sat: on. Number of scans: 5 total (1 pre and 4post-contrast scans, followed by a 5th scan at 5 minutes followingcontrast injection). All scans performed during suspended respiration,either at end expiration or end inspiration per standard institutionalpractice.

D. Clinical Trial Data: Day 5 Vascular Shutdown in Tumors and ColorectalCarcinoma Tumors after JX-594 Treatment

Acute vascular shutdown as measured by perfusion CT was previously seenin tumors treated directly with JX-594 by intratumoral injection (Liu etal., 2008). The inventors have applied DCE MRI analysis to followreduction in perfusion of tumors to follow the course of vascularshutdown and tumor necrosis in response to JX-594 treatment. Of the 16patients enrolled in a new clinical trial with optional DCE-MRI scanningon Day 5, 13 have received such scans, and there are additional examplesof vascular shutdown in patients with hepatocellular carcinoma (HCC)(FIG. 7).

In the HCC examples previously analyzed, it had appeared that directtumor injection was necessary to cause reduced tumor perfusion (FIG. 1,Liu et al., 2008). From this data, it was not predicted that vascularshutdown would occur in non-injected tumors in response to distantapplication of JX-594. Now, for the first time, the inventors show thatit is not necessary to inject every tumor to have this response, andthat distant, non-injected tumors can also show vascular shutdown (FIGS.8 and 9).

Furthermore, the inventors demonstrate that JX-594 can cause vascularshutdown in non-HCC tumors as evidenced in a patient with liver-basedmetastases of colorectal carcinoma (CRC), a tumor type considered lesswell-vascularized than HCC (FIG. 9), and therefore potentially lesslikely to incur vascular changes.

Example 1703

Patient 1703 had hepatocellular carcinoma and was enrolled in a Phase 2Clinical Trial of JX-594. JX-594 was injected into a single large tumor(10⁹ pfu/dose). After five days, DCE MRI showed acute vascular shutdown(top black and white panels)(FIG. 9). Bottom panels show an example ofsegmentation analysis used to quantify the extent of vascularshutdown/tumor necrosis (bottom panels)(FIG. 7).

Example 1708

Patient 1708 had hepatocellular carcinoma, with multiple tumors presentin the liver and was enrolled in a Phase 2 Clinical Trial of JX-594.JX-594 was injected into some but not all of the liver tumors (totaldose of 10⁸ pfu/dose). After five days, DCE MRI showed acutenecrosis/vascular shutdown in injected and non-injected liver-basedtumors. FIG. 8 shows two planes of view, including images both beforeand after JX-594 treatment.

Example 0204

Patient 0204 had colorectal carcinoma, with metastases present in theliver, lung and lymph nodes and was enrolled in a Phase 2 Clinical Trialof JX-594. JX-594 was injected into some but not all of the livermetastases (total dose of 10⁹ pfu/dose). After five days, DCE MRI showedacute necrosis/vascular shutdown in injected and non-injectedliver-based tumors. (FIG. 9)

E. Clinical Trial Data: JX-594 Potentiates Sorafenib and SutentAnti-VEGF Therapy

Five patients showing reperfusion on Week 8 after completing JX-594treatment subsequently received standard sorafenib dosing (400 mg twicedaily). Enhanced Choi responses were seen (Table A). These responseswere enhanced over any initial Choi response to JX-594 treatment alone(FIGS. 11, 12, and 13)

TABLE A Enhanced Choi Response in Patients treated with JX-594 followedby sorafenib. RECIST CHOI CHOI Dose Response to JX- Response to JX-response to Patient Level 594, Week 8 594, Week 8 sorafenib 1702 10⁹ SD(inj) PD Not evaluable Choi+ pfu/dose (new) 1705 10⁸ PD Choi+ −36% Choi+pfu/dose 1002 10⁹ SD (inj) PD Choi+ −121%  TBD pfu/dose (new) 1712 10⁹PD Choi+ −33% Choi+ pfu/dose 1713 10⁹ PD Choi− TBD pfu/dose

Example Sorafenib Only “Control Group” (Figure Included): Historically,the RECIST response rate to sorafenib alone is 1-2% (Llovet et al.,2008; Cheng et al., 2009). For a local control group assessed by Choicriteria, HCC patients that had not received JX-594, but had receivedsorafenib, were assessed for Choi response. In the hospital where 4 ofthe 5 JX-594 patients were treated, 26 other patients had receivedsorafenib in the same period. 7 patients died prior to responseassessment, and 15 patients were assessed for Choi response. Only 2 ofsorafenib-only patients showed a Choi+ response (26 total; 15 assessedfor Choi response). It should be noted that both of these patients hadreceived radiation therapy in conjunction with sorafenib therapy. Incomparison, the two patients who had received JX-594 therapy prior tosorafenib therapy during this period had a Choi+ response (FIG. 10), asurprising and extraordinary improvement in sorafenib effect on tumors.

In a sorafenib-only clinical trial which used MRI scans to assess tumornecrosis, some hepatic masses displayed central tumor necrosis, withmoderate increase in mean tumor necrosis from 9.8% at baseline to 27%after several courses of treatment (Abou-Alfa et al., 2006).

Example 1702

Patient 1702 had hepatocellular carcinoma and was enrolled in a Phase 2clinical trial for JX-594 and received three intratumoral doses ofJX-594 (10⁹ pfu/dose given two weeks apart). This patient was notevaluable for response to JX-594 using modified Choi criteria, howeverweek 8 scans showed stable disease (SD) in injected tumors butprogressive disease (PD) due to emergence of new tumors using modifiedRECIST criteria. Therefore, Patient 1702 went on to receive a standardcourse of Sorafenib treatment for 8 weeks (200 mg twice daily p.o.). DCEMRI scans taken at 4 and 8 weeks after initiation of Sorafenib treatmentshowed acute tumor necrosis. FIG. shows three different planes, withimages on the left from 4 weeks post-Sorafenib treatment and images onthe right from 8 weeks post-Sorafenib treatment. (FIG. 11)

Example 1705

Patient 1705 had hepatocellular carcinoma and was enrolled in a Phase 2Clinical Trial of JX-594. Five days after the first dose of JX-594, amarked reduction in perfusion by DCE MRI confirmed vascular shutdownoccurred in the tumors (top panels). After completing JX-594administration (three intratumoral doses of 10⁸ pfu/dose given two weeksapart), week 8 scans showed a response by modified CHOI criteria (−36%),but progressive disease (PD) by modified RECIST criteria. Therefore,Patient 1705 went on to receive a standard course of Sorafenib treatmentfor 4 weeks (400 mg twice daily p.o.). DCE MRI scans taken at 4 weeksshowed acute tumor necrosis (bottom right panel). (FIG. 12, FIG. 15,FIG. 16))

Example 1712

Patient 1712 had hepatocellular carcinoma and was enrolled in a Phase 2Clinical Trial of JX-594. After completing JX-594 administration (threeintratumoral doses of 10⁹ pfu/dose given two weeks apart), week 8 scansshowed a response by modified CHOI criteria (−33%), but progressivedisease (PD) by modified RECIST criteria. Patient 1712 received astandard course of Sorafenib treatment for 4 weeks (400 mg twice dailyp.o.). DCE-MRI scans taken at 4 weeks showed acute tumor necrosis. (FIG.13)

Example 11301 Sutent Patient

Sutent is another targeted cancer therapy approved for renal cellcarcinoma (RCC) that inhibits VEGF activity and angiogenesis. Patient11301 had RCC with metastases to the liver and was enrolled in a Phase 1Clinical Trial of JX-594 for treatment of liver-based tumors. Aftercompleting 2 courses of JX-594 administration (intratumoral doses of 10⁹pfu/dose given three weeks apart), week 6 assessment showed stabledisease by RECIST. The patient received two more courses of JX-594treatment yet one liver mass and a large (14 cm) abdominal massprogressed. Therefore, Patient 1705 went on to receive sutent therapy.Prognosis for the patient was poor based on low hemoglobin and extent ofliver metastases (Motzer et al., 2006). Surprisingly, a completeresponse in all of the patient's tumors followed (FIG. 14). Whole bodyPET scanning showed no signal. Survival post-JX-594 treatment is 3years+(patient is still alive). In comparison, historical RCC completeresponse rate to sutent alone in tumors greater than 10 cm is 0%. Lessthan 5% of RCC patients with poor prognosis have survival of 3 years.

Example JX16-HCC-03, IV+IT+IT+Sorafenib Patient: Patient JX16-HCC-03 hadhepatocellular carcinoma and was enrolled in a Phase 2 Clinical Trial ofJX-594. After completing JX-594 administration (one intravenous and twointratumoral doses) patient received sorafenib. DCE-MRI scans showedloss of perfusion 10 days after sorafenib initiation in a non-injectedextrahepatic tumor (FIG. 18)

* * *

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for treating cancer in a subjectpreviously administered a Wyeth strain vaccinia virus lacking afunctional thymidine kinase gene and expressing GM-CSF comprisingadministering an effective amount of an anti-angiogenic tyrosine kinaseinhibitor at least 1 week and up to 13 weeks after the vaccinia virustherapy, wherein the anti-angiogenic tyrosine kinase inhibitor issorafenib or sunitinib.
 2. The method of claim 1, wherein theanti-angiogenic tyrosine kinase inhibitor is administered afterdetermining whether a tumor is undergoing re-vascularization.
 3. Themethod of claim 2, wherein determining tumor re-vascularization is bynon-invasive imaging of the tumor.
 4. The method of claim 3, wherein thenon-invasive imaging is magnetic resonance imaging (MRI).
 5. The methodof claim 4, wherein the magnetic resonance imaging is dynamiccontrast-enhanced MRI (dce-MRI).
 6. The method of claim 1, wherein theanti-angiogenic tyrosine kinase inhibitor is administered at least 5, 6,7, or 8 weeks after the vaccinia virus administration.
 7. The method ofclaim 1, wherein the cancer is a brain tumor, a head & neck cancertumor, an esophageal tumor, a skin tumor, a lung tumor, a thymic tumor,a stomach tumor, a colon tumor, a liver tumor, an ovarian tumor, auterine tumor, a bladder tumor, a testicular tumor, a rectal tumor, abreast tumor, a kidney tumor, a pancreatic tumor, hepatocellularcarcinoma or a renal carcinoma.
 8. The method of claim 7, wherein thecancer is a hepatocellular carcinoma or a renal carcinoma.
 9. The methodof claim 7, wherein the tumor is a metastasis.
 10. The method of claim9, wherein the metastasis is colon or renal metastasis.
 11. The methodof claim 1, further comprising first administering to the subject thevaccinia virus.
 12. The method of claim 11, wherein the vaccinia virusis administered intratumorally, intravascularly, or both intravascularlyand intratumorally.
 13. The method of claim 1, wherein the vacciniavirus is JX-594.
 14. The method of claim 1, wherein the anti-angiogenickinase inhibitor is administered at least 2 weeks after the vacciniavirus administration.
 15. The method of claim 3, wherein thenon-invasive imaging is contrast enhanced CT scanning.